1. Field of the Invention
The invention relates to an apparatus (a microscope, respectively a microscopic system) and a method for obtaining a sub-resolution spatial information of one or more objects in a region of interest of an observed sample. In particular, the invention relates to the detection and localization of fluorescence molecules employed to label the measured objects.
2. Description of the Related Art
It is known that due to the wave character of the light the images in light microscopy are diffraction limited. This is manifested in that point-like objects are registered as blurred (Airy-) discs in the image space on the sensor. The function describing this blurring is referred to as Point-Spread-Function or shortly PSF. If the relation between the maximal resolution of a microscope and the used numerical aperture of the objective (NA) and the wavelength (Lambda):d_min=Lambda/(2×NA),which has been disclosed by Ernst Abbe (1873) is taken into account, one obtains a best resolution d_min of about 200 nm as the natural resolution limit when the best objectives and light in the visible region are used. Since numerous processes happen at a considerably smaller scale the overcoming of the Abbe-limit is one of the most difficult but the most important challenges in the modern light microscopy.
While Electron Microscopy and other ultrastructure imaging methods based on ionizing radiation have the great advantage of unprecedented resolution, “visible” light (i.e. light within the wavelength range of near ultraviolet to near infrared) offers other advantages, such as identification of multiple types of appropriately labeled molecules in single, three-dimensionally intact cells, and even in living ones. Thus, it is highly useful to complement the potential of ionizing radiation imaging procedures for the study of biological nanostructures with novel approaches to perform high resolution using visible light microscopy. Some examples of potential applications of this are in examining the nanostructure of cell membranes, or the genome structure of the cell nucleus.
For example, Fluorescence Energy Transfer (FRET) microscopy allows for example distance measurements between two molecule types down to the few nanometer level, using visible light for excitation. Fluorescence Correlation Spectroscopy or Fluorescence Recovery After Photobleaching (FRAP) make possible the analysis of intracellular mobilities or labeled, respectively marked molecules. For a full understanding of functional cellular processes, however, additional structural information is necessary. To solve these and many other important problems of cell biology and cellular biophysics, appropriate spatial analysis is indispensable. A serious problem in achieving this goal is that conventional light optical resolution is limited to about 200 nm laterally and 600 nm axially, meaning that cellular nanostructures cannot be adequately resolved to provide full functional information.
Various recently introduced laseroptical “nanoscopy” approaches permit to overcome this problem, and to extend the spatial analysis far beyond the “Abbe/Rayleigh Limit” of optical resolution (here assumed to correspond in the object plane (x,y) to about half the wavelength used, according to the original formulas, and to about one wavelength in the direction of the optical axis (z)). In particular in the fluorescence microscopy it was possible, predominantly in the last ten years, to steadily increase the achievable resolution of the diffraction limited systems.
Thus for example in the concept of confocal laser scanning fluorescence 4Pi-microscopy the object is scanned by laser light focused from all sided (“4Pi geometry”) and the fluorescence excited is detected “point-by-point”. Using two opposing high numerical aperture lenses to concentrate two opposing laser beams constructively in a joint focus, confocal laser scanning 4Pi-microscopy has become an established “nanoscopy” method, allowing an axial optical resolution down to the 100 nm range. This is a resolution 5-7 times better than what can be achieved by conventional fluorescence microscopy methods.
In many imaging applications, the structural information desired is the size of nanostructures which are separated from each other by a distance larger than the Abbe limit. To solve this problem, Spatially Modulated Illumination (SMI) far field light microscopy was developed as one of the many possibilities for using structured illumination to improve spatial analysis. SMI microscopy is based on the creation of a standing wave field of laser light, which can be realized in various ways, such as by focusing coherent light into the back focal planes of two opposing objective lenses of high numerical aperture. The fluorescently labeled object is placed between the two lenses and moved axially in small steps (e.g. 20 nm or 40 nm) through the standing wave field. At each step, the emitted fluorescence is registered by a high sensitive CCD camera. This procedure allows axial diameter measurements of individual fluorescent subwavelength sized objects down to few tens of nanometers, and also the determination of axial distances between “point-like” fluorescent objects (at lateral distances larger than the Abbe limit) down to the range of a few tens of nanometers and with a precision in the 1 nm range. Several biophysical application examples indicate the usefulness of SMI-microscopy for the study of the size of transcription factories and of individual gene regions.
The employment of a structured lighting and a structured detection increases the resolution of a far-field and confocal methods at about factor of two laterally and up to a factor of six along the optical axis (axially). The methods for improving the lateral resolution are often simple to integrate in existing microscopes or to be newly implemented. In contrast the methods for maximal improvement of the axial resolution mostly employ technically most complex optical paths. For the analysis of living systems the lateral methods, which are often applied as far-field methods, are sufficiently fast. The predominantly confocal methods for improvement of the axial resolution are scanning techniques and are as such either too slow or do not fulfill the high optical requirements as such measurements.
Stimulated Emmision Depletion (STED) microscopy is a focused beam method, in which the size of the excited region is greatly reduced by stimulated emission depletion. Presently, this technique allows an optical lateral ((x,y)) resolution in the 15-20 nm range using visible light. In cases where the field of view can be made sufficiently small (in the few nanometer range), in vivo STED microscopy with tens of frames/second has been reported.
STED Microscopy can be regarded as a special case of RESOLFT (reversible saturable optical fluorescence transition microscopy), where in principal, optical resolution in the few nm range should become possible using visible light.
RESOLFT-methods offer through exploitation of non-linear effects theoretically arbitrary high resolution down to molecular scale. The success of these methods is, however, restricted by the physical properties of the used dyes, respectively markers or labels and the optical properties of the observed sample, respectively slide preparation. A three-dimensional object reconstruction had to rely up till now on quite complicated methods employing structured illumination. With the currently used dyes it was possible to achieve a lateral and axial improvement of the resolution, however those methods were up till now not appropriate for imagining of active processes.
Based on the experience that “point like” fluorescent objects can be light microscopically localized with a precision of more than one order of magnitude better than the diffraction limited resolution of conventional far field fluorescence light microscopy, Spectral Precision Distance Microscopy (SPDM) was conceived about a decade ago.
SPDM is a far field light microscopy method based on:
                a) labelling of neighbouring “point like” objects with different spectral signatures (for abbreviation also called colors); and        b) spectrally selective registration to “sort” the emitted photons according to their spectral signature; and        c) high precision position monitoring.        
Originally the “different spectral signatures” were realized by different excitation/emission spectra, but were conceived to include also other “photon sorting” modes like fluorescence life times, photoluminescence and stochastic labelling schemes to allow photophysical discrimination. In combination with fluorescence life time measurements, the application of the SPDM concept to nanometer resolution of single molecules was experimentally confirmed. In particular, using fluorescent labels of different photostable spectral signatures and procedures involving in situ calibration of chromatic shifts, a lateral (2D) position and distance resolution below 30 nm, and a three dimensional (3D) resolution below 50 nm was achieved in fixed cell nuclei after specific FISH labeling of small DNA targets using a standard confocal laser scanning microscope. Thus, SPDM was successfully applied to analyze the supramolecular architecture of genome regions in intact 3D conserved cell nuclei by position and distance measurements considerably below the conventional, diffraction limited optical resolution of high numerical aperture far field fluorescence microscopes. The SPDM concept (also called “colocalization”) proved to be useful also in a variety of other applications, including single molecules.
During the first applications of the “SPDM” to biological nanostructure elucidation, the objects were labeled with fluorochrome molecules which had different emission wavelengths, and the signals were acquired synchronously. Fluorescence emission spectra of selectable molecules with typically 50 nm bandwidth are relatively broad. As such, in fluorescence microscopy, the detectable wavelengths are limited to a complete range of about 600 nm, and only a few “colors” (in the sense of variation in the emission spectra) can be used at the same time within the same object.
Since the original reports a number of conceptually related far field fluorescence methods have been proposed, for example BLINKING (meaning that the a light source emits light in pulses with a given frequency, much like the light houses); FPALM (fluorescence photoactivation localization microscopy), PALM (photoactivated localization microscopy), PALMIRA (PALM with independently running acquisition); STORM (stochastic optical reconstruction microscopy).
As a general denomination, all these approaches might be regarded as methods of “Spectrally Assigned Localization Microscopy” (SALM) where the localization of an object is assigned to a characteristic spectral signature. The underlying principle of these “SPDM/SALM” approaches is the “optical isolation” (in space and/or time domain) and hence independent localization of individual “point like” objects due to any photon based characteristics of the emitted light. This means that in a given diffraction limited observation volume defined for example by the (x,y, z) Full-Width-at-Half-Maxima (FWHM) of the Point-Spread-Function (PSF) of the microscope system used, at a given time interval and for a given spectral registration mode, only one such object (for example a singe molecule) or under certain conditions only few objects are registered.
By imaging fluorescent bursts of single molecules after light activation, the position of the molecules could be determined with a precision much higher than the full width at half maximum of the point spread function. In other words, these microscopy approaches are based on the registration of multiple (ex. thousands) of images of the same specimen, respectively same region of interest, so that the optical resolution is improved by “scanning” the fourth coordinate of the space-time continuum.
The molecules and proteins that are used for PALM and related techniques are fluorescent labels which are chemically modified (for example by adding appropriate side groups) in such a way that most of the fluorescent molecules are initially in an inactive state for the fluorescence excitation at a given wavelength λexc. This state (also called a “dark” spectral signature), can be changed to a fluorescent one (also called a “bright” spectral signature), for example by illumination with light of a defined wavelength λphot (for example in the near ultraviolet), which is different from the one of fluorescence excitation. If the activation of the fluorescent markers, or in other words the transition form a “dark” spectral signature to a “bright” spectral signature, is done stochastically using low intensities, only few molecules within one acquisition time interval, respectively acquisition time frame of the detector are activated and thus an optical isolation of their signals may be achieved. Due to subsequent illumination with λexc, the fluorescent signal emitted by these optically isolated molecules (“bright” spectral signature) is then registered until they are irreversibly bleached (i.e. until an irreversible transition to a “dark” spectral signature). From these fluorescent signals, the position of the single molecules can be determined with high precision. Under good optical conditions, localization accuracy in the few nm-range is possible. Repetition of this procedure (for example by registration of about 10,000 individual image frames) allows one to obtain the positions of the individual molecules even if their mutual distances are far below the Abbe/Rayleigh-limit. The photoactivation process at λphot and the use of a second laser line (λexc) can be avoided if an auto-activation of the molecules by a readout laser (λexc) is used.
The methods of the Spectrally Assigned Localization Microscopy (SALM)/Spectral-Precision-Distance Microscopy (shortly called thereafter localization microscopy) (DE 10052823.6, DE 29701663.3, U.S. Pat. No. 6,424,421, DE 19830596.6, U.S. Pat. No. 7,342,717, WO 2006/127692 A2, US 20080032414 A1) allow in principle a lateral resolution in a single-digit nanometer range. This localization microscopy is characterized in particular by the relatively low requirements concerning the needed optical and mechanical components. Also the adjustment of the apparatus is quite user-friendly. The localization microscopy uses for overcoming the Abbe-limit the fact, that one single fluorescent object can be almost arbitrary precisely localized. As described above, the precondition is that the diffraction limited disc corresponding to the fluorescence object is available spatially isolated, that is to say is not overlaid or superposed with other signals. The lateral position of the emitting molecule is generally determined from the center of the diffraction limited fluorescence disc. The precision with which such determination can be carried out is dependent on the number of the detected photons and on the related to it signal to noise ratio. Although the used sensors are typically two-dimensional sensor arrays, it is also possible to perform localization along the third space direction. Such localization requires, however, either very exact three-dimensional model functions of the point-spread-function (PSF) in combination with great number of the detected photons (>1000) or additional knowledge about the position of the observed molecule in the object space. To obtain such results is quite difficult using only methods of the pure localization microscopy, as disclosed for example in WO 2006/127692 A2. A method is also known that exploits astigmatism to empirically generate a three-dimensional point-spread-function, which is separated in axial and lateral portion and to fit this function to the collected data. The average precision, respectively accuracy with which the localization can be carried out is about 55 nm (23 nm standard deviation). This value represents a kind of natural limit for these methods due to the typical number of detected photons and the fact that the lens-PSF exhibits a higher blurring or smearing in axial direction. The pure localization microscopy with its single or multiple cyclic single point-reconstruction methods is ill-suited for the observation of active processes such as for example in vivo measurements of living cells. Accordingly it is always necessary to find a compromise between a sufficient number of detected photons and a realizable point density within one as small as possible time window. Also the mechanical stability of the microscope puts at very long detection times a further limit to the localization precision.
An object of the invention is to provide a method and an apparatus with which the limit of the localization precision, respectively accuracy of the up to now established localization microscopy can be overcome, while simultaneously accelerating and optimizing the localization process.